In this project the mechanisms of mutagenesis are investigated through the detailed study of its specificity. This is pursued by DNA sequencing of mutations in the Escherichia coli lacI gene, occurring either spontaneously or after treatment with a mutagen. The specificity of mutation provides a way to dissect the components of mutagenesis since different pathways produce characteristic patterns of mutation with regard to type of mutation and DNA sequence dependence. In order to understand the pathways that contribute to spontaneous mutagenesis, we have analyzed mutagenesis in E. coli wild type and several mutant derivatives affected in defined DNA replication and DNA repair pathways. This analysis provides insight into the role of the affected pathway in preventing mutagenesis. For example, mutagenesis in mutH, mutL or mutS strains, which are defective in postreplicative mismatch repair, has provided insight into the contribution of mismatch repair in preventing spontaneous mutations resulting from DNA replication errors. The spectrum of mutations in this strain also provides a detailed view on the nature of DNA replication errors as produced by the replication fork. Likewise, mutagenesis in a mutDmutL double mutator strain, defective in both mismatch repair and exonucleolytic proofreading, has been used to delineate the contribution of proofreading to in vivo replication fidelity. The combined data have allowed the derivation of the contributions of base selection, proofreading and postreplicative mismatch repair in vivo. We have also analyzed the possible contribution of DNA replication errors to spontaneous mutation. New E. coli mutants were isolated carrying a mutation in the dnaE gene and which replicate their DNA with increased fidelity (antimutator mutants). Analysis of spontaneous lacI mutants in one of these strains revealed a roughly two- fold reduction in overall frequency, suggesting that uncorrected DNA replication errors may comprise 50% of all spontaneous mutations in E. coli. The mutations that "disappear" in the antimutator strain are composed largely of transversions, suggesting (i) that transition replication errors are efficiently removed by DNA mismatch repair, and (ii) that spontaneous transition mutations likely result from sources other than replication errors, such as deamination and other types of DNA damage. We are currently investigating additional antimutator strains that may have differential specificities.